Selection of head and neck cancer patients for treatment with drugs targeting EGFR pathway

ABSTRACT

Methods using mass spectral data analysis and a classification algorithm provide an ability to determine whether a head and neck squamous cell carcinoma (HNSCC) patient is likely to benefit from a drug targeting an epidermal growth factor receptor pathway, including small molecule epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and monoclonal antibody EGFR inhibitors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of our prior U.S. patentapplication Ser. No. 11/396,328 filed Mar. 31, 2006, currently pending,published as U.S. patent publication No. 2007/0231921. The entirecontent of the '121 patent application publication is incorporated byreference herein.

BACKGROUND

This invention relates to the field of identifying cancer patients asbeing likely to benefit from treatment with drugs targeting theepidermal growth factor receptor (EGFR) pathway. The identification forinitial selection for treatment involves mass spectral analysis of bloodsamples from the patient in conjunction with a classification algorithmusing a training set of class-labeled spectra from other patients withthe disease.

Non-Small-Cell Lung Cancer (NSCLC) is a leading cause of death fromcancer in both men and women in the United States. There are at leastfour (4) distinct types of NSCLC, including adenocarcinoma, squamouscell, large cell, and bronchioalveolar carcinoma. Squamous cell(epidermoid) carcinoma of the lung is a microscopic type of cancer mostfrequently related to smoking. Adenocarcinoma of the lung accounts forover 50% of all lung cancer cases in the U.S. This cancer is more commonin women and is still the most frequent type seen in non-smokers. Largecell carcinoma, especially those with neuroendocrine features, iscommonly associated with spread of tumors to the brain. When NSCLCenters the blood stream, it can spread to distant sites such as theliver, bones, brain, and other places in the lung.

Treatment of NSCLC has been relatively poor over the years.Chemotherapy, the mainstay treatment of advanced cancers, is onlymarginally effective, with the exception of localized cancers. Whilesurgery is the most potentially curative therapeutic option for NSCLC,it is not always possible depending on the stage of the cancer.

Recent approaches for developing anti-cancer drugs to treat the NSCLCpatients focus on reducing or eliminating the ability for cancer cellsto grow and divide. These anti-cancer drugs are used to disrupt thesignals to the cells to tell them whether to grow or die. Normally, cellgrowth is tightly controlled by the signals that the cells receive. Incancer, however, this signaling goes wrong and the cells continue togrow and divide in an uncontrollable fashion, thereby forming a tumor.One of these signaling pathways begins when a chemical in the body,called epidermal growth factor, binds to a receptor that is found on thesurface of many cells in the body. The receptor, known as the epidermalgrowth factor receptor (EGFR) sends signals to the cells, through theactivation of an enzyme called tyrosine kinase (TK) that is found withinthe cells. The signals are used to notify cells to grow and divide.

The use of targeted therapies in oncology has opened new opportunitiesto improve treatment options in advanced stage solid tumors wherechemotherapy was previously the only viable option. For example, drugstargeting the epidermal growth factor receptor (EGFR) pathway (includingwithout limitation, Tarceva (erlotinib), Erbitux (cetuximab), Iressa(gefitinib)) have been approved or are in evaluation for treatment ofadvanced stage solid tumors in particular non-small cell lung cancer(NSCLC). Metro G et al, Rev Recent Clin Trials. 2006 January; 1(1):1-13.

One limitation of nearly all systemic cancer therapies is that a singleagent will be active in only a minority of patients. As the field oftargeted therapies evolves, it is becoming apparent that predictivebiomarkers are integral to the success of any given therapy. In fact,many agents that have been recently approved by the regulatoryauthorities have been in diseases that harbor a universal molecularalteration, and thus a de facto predictive marker (e.g. imatinib inchronic myelogenous leukemia), or in conjunction with an assay to selectpatients (e.g. trastuzumab in HER2 positive breast cancer). By the sametoken, administering a targeted agent to an unselected patientpopulation is usually accompanied by a modest to nonexistent responserate (e.g. gefitinib 250 mg in HNSCC). Ostensibly the successfuldevelopment of any drug should be linked to predictors of its efficacyas these markers would markedly increase the likelihood that anindividual patient will benefit. Given the morbidity and burden oftreating cancer patients with ineffective agents, it is imperative thatthese endeavors are undertaken.

While in some trials EGFR-Inhibitors (EGFR-I) have been shown togenerate sufficient survival benefit even in unselected populations, inothers there was no substantial benefit. This lead AstraZeneca towithdraw their EGFR-tyrosine kinase inhibitor (TKI) (gefitinib, Iressa)from the United States market. Even in the case of approved EGFR-Is ithas become more and more clear that efficient and reliable tests arenecessary to identify those patients that might benefit from treatmentwith EGFR-Is vs. those that are not likely to benefit. Ladanyi M, etal., Mod Pathol. 2008 May; 21 Suppl 2:S16-22.

In our prior U.S. patent application Ser. No. 11/396,328, published asU.S. patent publication No. 2007/0231921, we have shown that a simpleserum-based pre-treatment test using mass spectrometry and sophisticateddata analysis techniques using a classifier and a training set ofclass-labeled spectra from other patients with the disease has promisefor patient selection for treatment with drugs targeting the EGFRpathway in non-small cell lung cancer patients. See also Taguchi F. etal, JNCI 2007 v99(11), 838-846, the content of which is incorporated byreference herein. The test, called VeriStrat in its commercial version,assigns the label “VeriStrat good” or “VeriStrat poor” to pre-treatmentserum or plasma samples. It has been shown in the JNCI paper that“VeriStrat good” patients are more likely to benefit from EGFR-Itreatment than VeriStrat poor patients with a hazard ratio of “VeriStratgood” vs. “VeriStrat poor” patients of approximately 0.5.

Head and neck squamous cell carcinoma is used here to refer to a varietyof cancer characterized by squamous cell carcinomas of the oral cavity,pharynx and larynx, salivary glands, paranasal sinuses and nasal cavity,as well as the lymph nodes of the upper part of the neck. Head and neckcancers account for approximately 3 to 5 percent of all cancers in theUnited States. These cancers are more common in men and in people overage 50. Tobacco. (including smokeless tobacco) and alcohol use are themost important risk factors for head and neck cancers, particularlythose of the oral cavity, oropharynx, hypopharynx and larynx.Eighty-five percent of head and neck cancers are linked to tobacco use.

SUMMARY OF THE INVENTION

We have discovered that the methods of mass spectral analysis of patientsamples and classification using a training set described in our priorpatent application provide not only a selection tool for initiallyidentifying NSCLC patients as being likely to benefit from drugstargeting the EGFR pathway, but also that the methods provide aselection tool for selection of head and neck squamous cell carcinoma(HNSCC) cancer patients for treatment by such drugs.

We have further determined that the selection (including the selectionfor NSCLC patients and HNSCC patients) for such treatment applies to thetwo main classes of EGFR inhibitors (EGFR-I), namely (1) small moleculetyrosine kinase inhibitors (TKIs) such as getfitinib and erlotinib, and(2) monoclonal antibody EGFG-I such as cetuximab (Erbitux) andpanitumumab.

Additionally, as the methods of this disclosure require only simpleblood samples, the methods enable a fast and non-intrusive way ofselection of such patients.

In one specific embodiment, a method is disclosed of determining whethera HNSCC patient is likely to benefit from treatment with a drugtargeting the EGFR pathway (e.g., an EGFR-TKI such Tarceva (erlotinib);Erbitux (cetuximab), Iressa (gefitinib), or equivalent) comprising thesteps of:

a) obtaining a mass spectrum from a sample from the patient;

b) performing one or more predefined pre-processing steps on the massspectrum obtained in step a);

c) obtaining values of selected features in said spectrum at one or morepredefined m/z ranges after the pre-processing steps on the massspectrum in step b) have been performed;

d) using the values obtained in step c) in a classification algorithmusing a training set comprising class-labeled spectra produced fromsamples from other patients to identify the patient as likely to benefitwith treatment with the said drug.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart showing a method for selection of HNSCC cancerpatients for treatment with EGFR-I in accordance with a preferredembodiment of this invention.

FIG. 2 is a Kaplan-Meier plot for a set of HNSCC patients treated withgefitinib and the class label assigned to serum samples using the methodof FIG. 1. The plot indicates that patients labeled “good” had a betterprognosis following treatment with gefitinib than the patients labeled“poor”, with a hazard ratio of 0.41 (95% CI: 0.22-0.79) of good versuspoor.

FIG. 3 is a Kaplan-Meier plot for a set of HNSCC patients treated withcetuximab and the class label assigned to serum samples using the methodof FIG. 1. The plot indicates that patients labeled “good” had a betterprognosis following treatment with gefitinib than the patients labeled“poor”, with a hazard ratio of 0.26 (95% CI: 0.06-1.06) of good versuspoor.

DETAILED DESCRIPTION

We have examined the MS profiles from serum or plasma samples fromrecurrent and/or metastatic NSCLC and HNSCC patients who were treatedwith gefitinib, erlotinib or cetuximab as monotherapy, or combinationregimens containing the EGFR-I, as well as samples from patients whowere not treated with EGFR-I. The MALDI mass spectra were obtained fromeach sample and each patient was classified into “good” or “poor”outcome groups for survival comparison. We have found that the MSprofile was predictive of survival outcomes in all EGFRI-treatedcohorts, but not in the control cohorts.

The methods for selection of NSCLC and HNSCC patients for treatment withEGFR-I targeting is illustrated in flow chart form in FIG. 1 as aprocess 100.

At step 102, a serum or plasma sample is obtained from the patient. Inone embodiment, the serum samples are separated into three aliquots andthe mass spectroscopy and subsequent steps 104, 106 (including sub-steps108, 110 and 112), 114, 116 and 118 are performed independently on eachof the aliquots.

At step 104, the sample is subject to mass spectroscopy. A preferredmethod of mass spectroscopy is matrix assisted laser desorptionionization (MALDI) time of flight (TOF) mass spectroscopy, but othermethods are possible. Mass spectroscopy produces data points thatrepresent intensity values at a multitude of mass/charge (m/z) values,as is conventional in the art. In one example embodiment, the samplesare thawed and centrifuged at 1500 rpm for five minutes at four degreesCelsius. Further, the serum samples may be diluted 1:10, or 1:5, inMilliQ water. Diluted samples may be spotted in randomly allocatedpositions on a MALDI plate in triplicate (i.e., on three different MALDItargets). After 0.75 ul of diluted serum is spotted on a MALDI plate,0.75 ul of 35 mg/ml sinapinic acid (in 50% acetonitrile and 0.1%trifluoroacetic acid (TFA)) may be added and mixed by pipetting up anddown five times. Plates may be allowed to dry at room temperature. Itshould be understood that other techniques and procedures may beutilized for preparing and processing serum in accordance with theprinciples of the present invention.

Mass spectra may be acquired for positive ions in linear mode using aVoyager DE-PRO or DE-STR MALDI TOF mass spectrometer with automated ormanual collection of the spectra. Seventy five or one hundred spectraare collected from seven or five positions within each MALDI spot inorder to generate an average of 525 or 500 spectra for each serumspecimen. Spectra are externally calibrated using a mixture of proteinstandards (Insulin (bovine), thioredoxin (E. coli), and Apomyglobin(equine)).

At step 106, the spectra obtained in step 104 are subject to one or morepre-defined pre-processing steps. The pre-processing steps 106 areimplemented in a general purpose computer using software instructionsthat operate on the mass spectral data obtained in step 104. Thepre-processing steps 106 include background subtraction (step 108),normalization (step 110) and alignment (step 112). The step ofbackground subtraction preferably involves generating a robust,asymmetrical estimate of background in the spectrum and subtracts thebackground from the spectrum. Step 108 uses the background subtractiontechniques described in U.S. published applications 2007/0231921 andU.S. 2005/0267689, which are incorporated by reference herein. Thenormalization step 110 involves a normalization of the backgroundsubtracted spectrum. The normalization can take the form of a partialion current normalization, or a total ion current normalization, asdescribed in our prior patent application U.S. 2007/0231921. Step 112aligns the normalized, background subtracted spectrum to a predefinedmass scale, as described in U.S. 2007/0231921, which can be obtainedfrom investigation of the training set used by the classifier.

Once the pre-processing steps 106 are performed, the process 100proceeds to step 114 of obtaining values of selected features (peaks) inthe spectrum over predefined m/z ranges. Using the peak-width settingsof a peak finding algorithm, the normalized and background subtractedamplitudes may be integrated over these m/z ranges and assigned thisintegrated value (i.e., the area under the curve between the width ofthe feature) to a feature. For spectra where no peak has been detectedwithin this m/z range, the integration range may be defined as theinterval around the average m/z position of this feature with a widthcorresponding to the peak width at the current m/z position. This stepis also disclosed in further detail in our prior patent application U.S.2007/0231921.

At step 114, as described in our patent application published as US2007/0231921, the integrated values of features in the spectrum isobtained at one or more of the following m/z ranges:

5732 to 57955811 to 58756398 to 646911376 to 1151511459 to 1159911614 to 1175611687 to 1183111830 to 1197612375 to 1252923183 to 2352523279 to 23622 and65902 to 67502.

In a preferred embodiment, values are obtained at least eight of thesem/z ranges, and more preferably at all 12 of these ranges. Thesignificance, and methods of discovery of these peaks, is explained inthe prior patent application publication U.S. 2007/0231921.

At step 116, the values obtained at step 114 are supplied to aclassifier, which in the illustrated embodiment is a K-nearest neighbor(KNN) classifier. The classifier makes use of a training set of classlabeled spectra from a multitude of other patients (NSCLC or HNSCCcancer patients). The application of the KNN classification algorithm tothe values at 114 and the training set is explained in our patentapplication publication U.S. 2007/0231921. Other classifiers can beused, including a probabilistic KNN classifier or other classifier.

At step 118, the classifier produces a label for the spectrum, either“good”, “poor” or “undefined”. As mentioned above, steps 104-118 areperformed in parallel on three separate aliquots from a given patientsample. At step 120, a check is made to determine whether all threealiquots produce the same class label. If not, an undefined result isreturned as indicated at step 122. If all aliquots produce the samelabel, the label is reported as indicated at step 124.

If the label reported at step 124 is “good” it indicates that thepatient is likely to benefit from administration of the EGFR pathwaytargeting drug, or continued administration in the case of monitoring apatient in the course of treatment. If the label reported at step 124 is“poor” it indicates that the patient is not likely to benefit fromtreatment by such a drug.

It will be understood that steps 106, 114, 116 and 118 are typicallyperformed in a programmed general purpose computer using software codingthe pre-processing step 106, the obtaining of spectral values in step114, the application of the KNN classification algorithm in step 116 andthe generation of the class label in step 118. The training set of classlabeled spectra used in step 116 is stored in memory in the computer orin a memory accessible to the computer.

The methods described in this application have been applied to a set of55 samples from HNSCC patients that were collected before treatment withgefitinib (tradename Iressa, AstraZeneca). Of these 31 yielded the label“good”, 23 yielded the label “poor”, and 1 resulted in the label“undefined”. The analysis was performed in a fully blinded manner, i.e.no clinical data were available during the determination of the label.Once the labels were generated the clinical data were unblinded and aKaplan-Meier analysis for overall survival could be performed from theclinical data for the endpoint overall survival. The Kaplan-Meier curvesare shown in FIG. 2 for the patients labeled “good” and “poor”. Thepatients labeled “good” had a better prognosis following treatment withgefitinib than the patients labeled “poor” with a hazard ratio of 0.41(95% CI: 0.22-0.79) of good versus poor. The good and poor curves arestatistically significantly different with a log-rank p-value of 0.007.These results indicate that the test described in this application canbe used to separate HNSCC patients into groups with statisticallydifferent prognosis following treatment with gefitinib.

The methods described in this application have also been applied to aset of 21 samples from HNSCC patients that were collected beforetreatment with cetukimab (tradename Erbitux, Imclone). Of these 16yielded the label “good” and 5 yielded the label “poor”. The analysiswas performed in a fully blinded manner, i.e. no clinical data wereavailable during the determination of the label. Once the labels weregenerated the clinical data were unblinded and a Kaplan-Meier analysisfor overall survival could be performed from the clinical data for theendpoint overall survival. The Kaplan-Meier curves are shown in FIG. 3for the patients labeled “good” and “poor”. The patients labeled “good”had a better prognosis following treatment with cetuximab than thepatients labeled “poor” with a hazard ratio of 0.26 (95% CI: 0.06-1.06)of good versus poor. The good and poor curves are close to statisticallysignificantly different with a log-rank p-value of 0.061. These resultsindicate that the test described in this application can be used toseparate HNSCC patients into groups with statistically differentprognosis following treatment with cetuximab.

From the above discussion, it will be appreciated that we have describeda method of determining whether a HNSCC patient is likely to benefitfrom treatment with a drug targeting the EGFR pathway, comprising thesteps of:

a) obtaining a mass spectrum from a sample from the patient;

b) performing one or more predefined pre-processing steps on the massspectrum obtained in step a);

c) obtaining values of selected features in said spectrum at one or morepredefined m/z ranges after the pre-processing steps on the massspectrum in step b) have been performed; and

d) using the values obtained in step c) in a classification algorithmusing a training set comprising class-labeled spectra produced fromsamples from other patients to identify the patient as being likely tobenefit from treatment with the said drug.

In preferred embodiments, the one or more m/z ranges comprises one ormore m/z ranges selected from the group of m/z ranges consisting of:

5732 to 57955811 to 58756398 to 646911376 to 1151511459 to 1159911614 to 1175611687 to 1183111830 to 1197612375 to 1252923183 to 2352523279 to 23622 and65902 to 67502.

Preferably but not necessarily, the mass spectrum is obtained from aMALDI mass spectrometer.

The drug for which the patient is identified as being likely to benefitfrom may comprise a small molecule epidermal growth factor receptortyrosine kinase inhibitor, such as getfitinib or erlotinib, oralternatively a monoclonal antibody epidermal growth factor receptorinhibitor such as cetuximab.

Variations from the particular details of the preferred embodimentsdisclosed are of course possible without departure from the scope of theinvention. All questions of scope are to be determined by reference tothe appended claims.

1. A method of determining whether a head and neck squamous cellcarcinoma (HNSCC) patient is likely to benefit from treatment with adrug targeting the EGFR pathway, comprising the steps of: a) obtaining amass spectrum from a sample from the patient; b) performing one or morepredefined pre-processing steps on the mass spectrum obtained in stepa); c) obtaining values of selected features in said spectrum at one ormore predefined m/z ranges after the pre-processing steps on the massspectrum in step b) have been performed; and d) using the valuesobtained in step c) in a classification algorithm using a training setcomprising class-labeled spectra produced from samples from otherpatients to identify the patient as being likely to benefit fromtreatment with the said drug.
 2. The method of claim 1, wherein the oneor more m/z ranges comprises one or more m/z ranges selected from thegroup of m/z ranges consisting of: 5732 to 5795 5811 to 5875 6398 to6469 11376 to 11515 11459 to 11599 11614 to 11756 11687 to 11831 11830to 11976 12375 to 12529 23183 to 23525 23279 to 23622 and 65902 to67502.
 3. The method of claim 1, wherein the mass spectrum is obtainedfrom a MALDI mass spectrometer.
 4. The method of claim 1, wherein thedrug comprises a small molecule epidermal growth factor receptortyrosine kinase inhibitor.
 5. The method of claim 1, wherein the drugcomprises a monoclonal antibody epidermal growth factor receptorinhibitor.